Described herein are bivalent binding molecules that can activate or inhibit 7 transmembrane G protein-coupled receptors. Also described herein are methods for identifying and preparing bivalent binding molecules to 7 transmembrane G protein-coupled receptors. The methods disclosed herein are based on the SELEX method for generating high affinity nucleic acid ligands, also termed aptamers. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The bivalent binding molecules of this invention comprise two or more binding domains to two or more different epitopes of the same 7 transmembrane G protein-coupled receptor. In a preferred embodiment, at least one binding domain is an aptamer. These bivalent binding molecules are useful as therapeutic and diagnostic agents.
The seven transmembrane receptors (also known as G protein-coupled receptors or 7TM G protein-coupled receptors) comprise a superfamily of structurally related integral proteins. 7TM G protein-coupled receptors exhibit detectable amino acid sequence similarity and all appear to share a number of structural features (See, FIG. 1). These features include: an extracellular amino terminus (EAT); seven predominantly hydrophobic alpha-helical domains (of about 20-30 amino acids) which are believed to span the cell membranes and are referred to as transmembrane domains (TMD 1-7); six loops which connect the transmembrane domains (three extracellular loops (ELs) and three intracellular loops (ILs)); and a cytoplasmic carboxy terminus (CCT).
Each 7TM G protein-coupled receptor is predicted to associate with a particular heterotrimeric G protein (composed of xcex1, xcex2 and xcex3 subunits) at the intracellular surface of the plasma membrane. Upon binding of an agonist to the receptor, a conformational change occurs in the receptor, which enables interaction of the intracellular loops of the receptor with its associated intracellular, membrane-anchored heterotrimeric G protein. This causes the alpha-subunit of the G protein to exchange a bound GDP molecule for a GTP molecule and to dissociate from the xcex2 and xcex3 subunits. The GTP-bound form of the alpha-subunit in turn stimulates specific intracellular signal-transducing enzymes and channels.
It has been proposed that 7TM G protein-coupled receptors adopt two major conformations: an active, G protein-coupled and thus transducing conformation and an inactive (non-transducing) conformation (Schwartz, T. W. et al., Cur. Pharmaceut. Design, 1:325-342 (1995)). The binding of an agonist or antagonist selectively stabilizes the active and the inactive receptor conformations, respectively, as predicted by the allosteric regulation of proteins as suggested by Monod, Wymann and Changeux (J. Mol. Biol., 2:7439-7442 (1965)). Agonists are thus extracellularly acting allosteric ligands that increase the signal transduction rate at intracellular sites upon binding. Antagonists are extracellularly acting ligands that inhibit signal transduction upon binding.
The 7TM G protein-coupled receptors are the largest family of cell-surface receptors comprising several hundred distinct receptors, and over 100 receptors have been cloned. The transmembrane segments of 7TM G protein-coupled receptor family members exhibit considerable homology, whereas the extracellular connecting loops are less conserved, showing high homology only between closely related receptor subtypes. The 7TM G protein-coupled receptors can be grouped based on their homology levels and/or the nature of the ligands they recognize. For example, the interleukin-8 receptor, the angiotensin II receptor, the thrombin receptor, the endothelin receptors, the N-formyl peptide receptor and the C5a receptor all bind peptide ligands and share 20-40% amino acid similarity.
The 7TM G protein-coupled receptors bind a wide variety of ligands of different molecular size ranging from small monoamines and other small molecules, to large neurotransmitters and peptide hormones. The family of 7TM G protein-coupled receptors also includes the receptors for light (rhodopsin), for odors (olfactory receptors) and for taste (gustatory receptors). Additionally, the conserved structure among 7TM G protein-coupled receptors has allowed for the cloning of many novel genes encoding 7TM G protein-coupled receptors whose natural ligand and function are yet to be elucidated. These receptors are referred to asxe2x80x9corphanxe2x80x9d receptors. Table 1 lists a number of 7TM G protein-coupled receptors which have been cloned and expressed.
Because of the involvement of 7TM G-protein-coupled receptors in the regulation of many critically important biological functions and disease conditions, many of these functions and conditions may be influenced or determined by the state of activation or inhibition (e.g., blockade) of a 7TM G protein-coupled receptor. However, these receptors are difficult to purify. The proteins can be removed from the membrane only by the action of detergents, which denatures some proteins. In addition, most membrane proteins are not soluble in water. To date, few novel agonists or antagonists to these receptors have been identified. Common methods have involved generating antibodies to 7TM G protein-coupled receptors expressed in cells which have been administered to a host. Lerner et al. (PCT Application No. WO 98/03632) have described peptide dimer agonists for 7TM G protein-coupled receptors. These dimers were comprised of two known peptide agonists or antagonists (e.g., natural ligands) to different 7TM G protein-coupled receptors.
It would be useful to be able to develop agonists and antagonists to the specific binding portions of 7TM G protein-coupled receptors. Attempts to achieve expression of only the ligand binding portion of a 7TM G protein-coupled receptor have been unreproducible or have resulted in inefficient and/or unpredictable levels of expression (Xie, U. B., et al, J. Biol. Chem. 265:21441-21420 (1990); Tsai-Morris, C. H., et al. J. Biol. Chem. 265:19385-19388 (1990)).
As suggested in Lemer et al., bivalent binding molecules can have utility as therapeutics. More specifically, bivalent and bispecific antibodies have many practical applications, including in immunodiagnosis and therapy. Bivalency can allow antibodies to bind to multimeric antigens with great avidity; multivalency theoretically can increase apparent binding affinity by several orders of magnitude (Crothers, D. M. et al., Immunochemistry 9: 341-351 (1972)). Bispecificity can allow the cross-linking of two antigens, for example, in recruiting cytotoxic T cells to mediate killing of a tumor cell. Specific examples of bivalent molecules capable of binding to adjacent epitopes include small bivalent antibodies composed of either antibody fragments (Fab) or single chain antibodies (Fv) (Pack, P. et al., Biochemistry 31, 1579-1584 (1992); Holliger, P. et al., Proc. Natl. Acad. Sci. USA 9, 6444-6448 (1993); Mallender, W. D. et al., J. Biol. Chem., 269: 199-206 (1994)). Neri, D. et al. (J. Mol. Biol., 246:367-373 (1995)) developed a bispecific antibody fragment, binding two antibodies with a polypeptide chain, that recognizes adjacent and non-overlapping epitopes of lysozyme and is able to bind both epitopes simultaneously.
Bivalent peptides, such as receptor-adhesive modular proteins (xe2x80x9cRAMPsxe2x80x9d), have been used in an alternative approach to cell targeting. M. Engel et al., (Biochemistry 30: 3161-3169 (1991)) and C. A. Slate et al., (Int. J. Peptide Protein Res. 45: 290-298 (1995)) have designed large synthetic peptides, which contain two ligand sites separated by a spacer region and a dimerization domain.
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential Enrichment, termed the SELEX process, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment,xe2x80x9d now abandoned; U.S. Pat. application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,475,096; U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cNucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is herein specifically incorporated by reference. These applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structurexe2x80x9d, xe2x80x9cnow abandoned, (see, U.S. Pat. No. 5,707,796)xe2x80x9d abandoned in favor of U.S. Ser. No. 08/198,670, now U.S. Pat. No. 5,707,796, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligandsxe2x80x9d, abandoned in favor of U.S. Ser. No. 08/443,959, filed May 18, 1995, wich was abandoned in favor of U.S. Ser. No. 08/612,895, filed Sep. 16, 1994, now U.S. Pat. No. 5,763,177, describes a SELEX-based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeinexe2x80x9d, now U.S. Pat. No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEXxe2x80x9d, xe2x80x9cnow abandoned, (see, U.S. Pat. No. 5,567,588)xe2x80x9d abandoned in favor of U.S. Ser. No. 08/461,069, filed Jun. 5, 1995, now U.S. Pat. No. 5,567,588, and U.S. patent application Ser. No. 08/792,075, filed Jan. 31, 1997, entitled xe2x80x9cFlow Cell SELEXxe2x80x9d, now U.S. Pat. No. 5,861,254, describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled xe2x80x9cNucleic Acid Ligands to HIV-RT and HIV-1 Revxe2x80x9d, now U.S. Pat. No. 5,496,938, describes methods for obtaining improved nucleic acid ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEXxe2x80x9d, now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotidesxe2x80x9d now U.S. Pat. No. 5,861,254, xe2x80x9cnow abandoned, (see, U.S. Pat. No. 5,660,985)xe2x80x9d with abandoned in favor of U.S. Ser. No. 08/430,709, now U.S. Pat. No. 5,660,986, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, now U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacementxe2x80x9d, now U.S. Pat. No. 5,756,703, describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEXxe2x80x9d, now U.S. Pat. No. 5,637,459 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Blended SELEXxe2x80x9d, now U.S. Pat. No. 5,683,867, respectively. The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic or Non-Immunogenic, High Molecular Weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed, May 4, 1995, entitled xe2x80x9cNucleic Acid Ligand Complexes now U.S. Pat. No. 6,011,020xe2x80x9d. VEGF Nucleic Acid Ligands that are associated with a Lipophilic Compound, such as diacyl glycerol or dialkyl glycerol, in a diagnostic or therapeutic complex are described in U.S. patent application Serial No. 08/739,109, filed Oct. 25, 1996, entitled xe2x80x9cVascular Endothelial-Growth Factor (VEGF) Nucleic Acid Ligand Complexes now U.S. Pat. No. 5,839,228xe2x80x9d. VEGF Nucleic Acid Ligands that are associated with a Lipophilic Compound, such as a glycerol lipid, or a Non-Immunogenic, High Molecular Weight Compound, such as polyalkylene glycol, are further described in U.S. patent application Ser. No. 08/897,351, filed Jul. 21, 1997, now U.S. Pat. No. 6,051,698 entitled xe2x80x9cVascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexesxe2x80x9d. VEGF Nucleic Acid Ligands that are associated with a non-immunogenic, high molecular weight compound or lipophilic compound are also further described in PCT Application Publication No. WO 98/18480, filed Oct. 17, 1997, entitled xe2x80x9cVascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexesxe2x80x9d. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
The identification of nucleic acid ligands to small, flexible peptides via the SELEX method has been explored. Small peptides have flexible structures and usually exist in solution as an equilibrium of multiple conformers, and thus it was initially thought that binding affinities may be limited by the conformational entropy lost upon binding a flexible peptide. However, the feasibility of identifying nucleic acid ligands to small peptides in solution was demonstrated in U.S. Pat. No. 5,648,214, filed Sep. 9, 1994, entitled xe2x80x9cHigh-Affinity Oligonucleotide Ligands to the Tachykinin Substance Pxe2x80x9d, which is incorporated herein by reference. In this patent, high affinity RNA nucleic acid ligands to substance P, an 11 amino acid peptide were identified.
Xu and Ellington (Proc. Natl. Acad. Sci. USA, 93:7475-7480 (1996)) employed the human immunodeficiency virus type 1 (HIV-1) Rev to further explore how peptide and protein epitopes are recognized by nucleic acid ligands. In this study, RNA nucleic acid ligands were selected to bind to the isolated Rev34-50 peptide. It was observed that RNA nucleic acid ligands could not only recognize the sequence of this peptide, but that these nucleic acid ligands could also bind the corresponding native epitope on the Rev protein, albeit with lower affinity.
The present invention provides bivalent binding molecules comprising two or more binding domains which bind simultaneously to two or more epitopes of the same 7TM G protein-coupled receptor and thus increase the binding affinity relative to the binding of a single binding domain. The binding domains are identified using synthetic peptides corresponding to all or a portion of the extracellular binding domains and therefore purified and isolated receptor proteins are not required.
The present invention describes bivalent binding molecules having binding affinity for two or more epitopes of the same 7TM G protein-coupled receptor and methods for generating and producing such bivalent binding molecules. The bivalent binding molecules described herein may be agonists, antagonists or superagonists. In one embodiment the bivalent binding molecules of the invention comprise a first binding domain coupled to a second binding domain, the first and second binding domains being ligands to a first and second epitopes. In preferred embodiments, the first and second epitopes are located on different extracellular loops of the same 7TM G protein-coupled receptor. In a preferred embodiment, at least one binding domain of the bivalent binding molecule is an aptamer. In one embodiment, the first and second binding domains are coupled via a linker.
In a preferred embodiment, both the first and second binding domains are aptamers to first and second epitopes of two different extracellular loops. In this embodiment, the aptamers are coupled to each other at either their 5xe2x80x2 or 3xe2x80x2 ends. In a preferred embodiment, the aptamers are SELEX-derived aptamers.
In another embodiment, the first binding domain is an aptamer to a first epitope of a 7TM G protein-coupled receptor, and the second binding domain is a non-aptamer binding domain that binds to a second epitope of the same 7TM G protein-coupled receptor.
In one embodiment of the method of this invention, a method for identifying bivalent binding molecules to 7TM G protein-coupled receptors is described, wherein the bivalent binding molecules comprise two binding domains, each of which is an aptamer. In this method, the bivalent binding molecules are identified generally by applying the Chimeric SELEX methods described in U.S. Pat. No. 5,637,459, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEXxe2x80x9d, which is incorporated herein by reference.
In another embodiment of the method of this invention, a method for identifying bivalent binding molecules to 7TM G protein-coupled receptors is described, wherein one binding domain of the bivalent molecule is an aptamer and the other is a non-aptamer binding domain. In this method, the bivalent compounds are identified generally by applying the Blended SELEX methods described in U.S. Pat. No. 5,683,867, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Blended SELEXxe2x80x9d, which is incorporated herein by reference. Suitable non-aptamer binding domains include all or a portion of a natural ligand to the 7TM G protein-coupled receptor.
In another embodiment of the method of this invention, a method for identifying bivalent binding molecules to 7TM G protein-coupled receptors is described, wherein both binding domains are unnatural L-aptamers (mirror images of the natural D-aptamers). In other embodiments, the first binding domain is an aptamer of non-natural handedness to a first epitope and the second binding domain is either an aptamer of natural handedness or a non-aptamer binding domain to a second epitope.
This invention further includes the bivalent binding molecules to 7TM G protein-coupled receptors generated by the methods of this invention. These bivalent binding molecules may contain one or more modified nucleotides such as nucleotides modified at the 2xe2x80x2- and/or 5 and/or 8 positions. Such modifications include but are not limited to nucleotides containing 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F) and 2xe2x80x2-O-methyl (2xe2x80x2-Oxe2x80x94Me) groups.
With certain 7TM G protein-coupled receptors, it may not be necessary for a binding ligand to be bivalent. Therefore this invention further includes aptamers to 7TM G protein-coupled receptors identified by the SELEX process which can act as agonists or antagonists of the receptor.
This invention further provides a method for treating diseases or conditions mediated by 7TM G protein-coupled receptors.